PEGylation is the process whereby polyethylene glycol (PEG) is covalently bonded to a drug molecule in order to improve its pharmacokinetic, pharmacodynamic, and/or immunological characteristics. PEG is a water-soluble, non-toxic, non-immunogenic polymer approved by the FDA for internal use. In general, PEGylation increases drug solubility and reduces immunogenicity. PEGylation also increases drug stability and retention in blood, and it reduces proteolysis and renal extraction; these in turn enable reduced dosing frequency, leading to reduced costs and improved quality of life and compliance for the patient. To date, PEGylation has been used primarily to modify therapeutic proteins, though it has also been applied to peptides and antibody fragments, as well as small molecule drugs.
Structural characterization of drug molecules is essential for successful regulatory approval. Proper characterization of PEGs and PEGylated compounds requires confirmation of end group structure, mass of the repeat unit, average molecular weight, and molecular weight distribution/polydispersity. The accurate determination of these properties can be a formidable analytical challenge, in large part because of the heterogeneity/polydispersity of PEG, but also because of the high mass of the molecules in question, often ≧40 kDa. For a peptide or protein drug, it is also necessary to determine if the PEGylation reaction results in unwanted modifications to amino acid side chains. Moreover, the quality of the PEGylated product following purification must be evaluated to ensure the process is capable of yielding material of appropriate purity, and the stability of the PEGylated molecule under formulation conditions and during long term storage must be demonstrated. Similarly, the quality of the PEG starting material used in the PEGylation reaction must be assessed as it directly affects the final drug product.
Mass spectrometry (MS) is becoming an increasingly important technique for the structural characterization of polymers, including PEGs and PEGylated compounds. Matrix assisted laser desorption ionization (MALDI) combined with time-of-flight (TOF) mass analysis has been used most often for characterization of polymers by mass spectrometry. However, the structural information provided by MALDI-TOF-MS can be inadequate for larger peptides or proteins, such as those modified with larger PEGs (e.g. >20 kDa). This is because MALDI predominantly generates singly-charged ions while both the resolving power and the detection efficiency of TOF-MS instruments decrease with increasing mass to charge ratio. The singly charged ions generated with MALDI from large peptides or proteins can therefore be difficult to resolve and detect.
Electrospray ionization (ESI) may be used with TOF-MS for the analysis of polymeric compounds. ESI is different from MALDI in that it normally yields multiply charged ions distributed over a range of charge-states. Due to the multiple-charging, the ions of even very large species have mass to charge ratio values suitable for resolution and detection with TOF-MS. There is, however, a major problem with using ESI for the analysis of large polymers. That is, the peak envelope due to the distribution of molecular masses for a given charge-state generally overlaps with the peak envelope for the adjacent charge-state(s). The resulting spectra are congested and often uninterpretable, and thus incapable of yielding the desired structural information. In order to overcome this problem it is known to perform supplemental charge stripping on the electro-sprayed ions, thus lowering their charge-states and separating the peak envelopes.
Known methods of charge stripping involve generating reagent ions that are oppositely charged to the electro-sprayed ions and then reacting these reagent ions with the electro-sprayed ions in order to reduce the charge states of the electro-sprayed ions. Some of these known methods use radioactive sources to generate the reagent ions. The use of radioactive material is undesirable for obvious reasons, but it also has inherent stability issues stemming from the natural decay of radioactivity. Furthermore, in known charge stripping instruments the highly charged electrified sprayer of the ESI ion source interferes with the oppositely charged reagent ions and affects the charge reduction reactions.
It is therefore desired to provide an improved method of mass spectrometry or ion mobility spectrometry.